Identification of the domains in SET responsible for binding Sgo1. (A) Schematic drawing of the functional domains of human SET. The residues that are important for the Sgo1–SET interaction were labeled, and the red ones were chosen for the subsequent studies. (B and C) Triple mutations (3K) of D56K, E60K, and E64K abolish Shugoshin–SET interactions in vitro. In vitro–translated S35-radiolabeled human Sgo1 (B) and human Sgo2 (C) were incubated with recombinant GST-SET (full length) WT or 3K proteins. GST bead–pelleted proteins were resolved with SDS-PAGE and stained with Coomassie blue. Dried gels were visualized by a phospho-imager. (D) Triple mutations (3K) of D56K, E60K, and E64K abolish the Sgo1–SET interaction in cells. Lysates of mitotic HeLa Tet-On cells transfected with vectors (V) or plasmids containing Myc-Sgo1 (1–472) and GFP-SET WT or 3K (D56K/E60K/E64K in SET-α) were incubated with antibody against GFP. The immunoprecipitated proteins with distinct doses were resolved with SDS-PAGE and blotted with the indicated antibodies. (E and F) Triple mutations (3K) of D56K, E60K, and E64K barely affect SET binding to PP2A and histones. Lysates of nocodazole-arrested HeLa Tet-On cells transfected with vectors (V) or plasmids containing GFP-SET WT, 3K, or SET ΔC (truncation of the region 227–290 in SET-α; E) were treated with anti-GFP antibody. The immunoprecipitated proteins were resolved with SDS-PAGE and blotted with the indicated antibodies. Control (Ctl) in F denotes mock transfection without vectors. (G) Triple mutations (3K) of D56K, E60K, and E64K do not affect SET dimerization. Lysates of nocodazole-arrested HeLa Tet-On cells transfected with vectors (Ctl) or plasmids containing GFP-SET (WT or 3K) and Myc-SET (WT or 3K) were incubated with antibody against Myc. The pelleted proteins were resolved with SDS-PAGE and blotted with the indicated antibodies.